The goal of this project, as part of the multi-project application, is the rational design of a tetravalent epitope-defined genetic dengue vaccine. This research will focus on the analyses of ex vivo human immune responses of patients of a well-characterized dengue cohort (Core B).to predicted HLA promiscuous class I and II T-cell peptide epitopes (Project 1) and B-cell epitopes (Project 4). Initially, the immunogenicity of the putative T-cell epitopes will be tested by ELISPOT assay using synthetic peptides to stimulate the cellular responses of dengue patients. The confirmed immunogenic epitopes will be analyzed for associations to specific HLA allotypes, virus infection serotype/genotype history, severity of the disease, level of dengue virus infected peripheral blood mononuclear cells, as measured by a quantitative RT-PCR, specific for the replicative viral genome template (Project 3) and other relevant parameters. Validated T-cell epitopes of all four dengue serotypes will be selected for their correlation with more favorable outcomes and combined with neutralizing B-cell epitopes (Project 4) into a MIIC (major histocompatibility complex class II compartment) targeted chimeric gene construct, containing the lysosomal trafficking signals from the lysosomal associated membrane protein, LAMP (Project 4). The immunogenicity of candidate dengue LAMP/multi-epitope chimeric vaccine constructs will be tested in human tissues ex vivo. The model system for evaluation of the dengue LAMP/multi-epitope vaccine will test for T-cell epitope processing, presentation and immunogenicity carried out by monocyte derived dendritic cells (MDDC) transfected by the genetic vaccine encoding the dengue epitopes as LAMP chimeras. These MDDC will be used as antigen presenting cells to stimulate the patient-specific T-cells. The immune responses to the candidate dengue LAMPtmulti-epitope vaccine chimeras will then be studied for its clinical correlations and evaluation of the breath of its HLA responses. The dengue LAMP/multi-epitope chimeric vaccine design is intended to include all the epitopes needed to elicit responses by the majority of the HLA alleles and its responses to be correlated with lower rates of cellular virus infection and better clinical outcomes. In addition, culture supernatants of the cellular responses will be used to search for novel biological markers of vaccine efficacy and toxicity, which may be useful as markers for the clinical development of dengue vaccines. The final LAMP/multi-epitope chimeric vaccine formulation would be ready to be inserted into many gene delivery vehicles (DNA, AAV, Adeno, Lentivirus, Vaccinia, etc.) and used in clinical studies.